Porous membrane enabled mass spectrometry characterization of microfluidic devices

ABSTRACT

A system for sampling a liquid includes a sample fluid conduit including a membrane having pores. The membrane prevents the passage of the sample liquid through the pores at a first pressure of the sample liquid in the sample fluid conduit. A surface sampling capture probe has a distal end. The capture probe includes a solvent supply conduit and a solvent exhaust conduit. A solvent composition flowing at the distal end of the capture probe establishes a liquid junction with the membrane and establishes a second pressure within the liquid junction at the membrane. The second pressure is lower than the first pressure. Sample liquid will be drawn through the pores of the membrane by the second pressure at the liquid junction. A method for sampling a liquid and for performing chemical analysis on a liquid are also disclosed.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No.DE-AC05-00OR22725 awarded by the U.S. Department of Energy. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to mass spectrometry, and moreparticularly to the mass spectrometry analysis of analytes in liquidsamples.

BACKGROUND OF THE INVENTION

The chemical analysis of living systems has become more feasible withthe development of sensitive analytical techniques. Mass spectrometriccharacterization requires destruction of the system being analyzed,negating the ability to measure systems in situ especially for smallsystems in fluid media. Most applications of in situ mass spectrometryhave been directed toward the chemical analysis of produce, leaves,skin, or otherwise stable, non-dynamic samples. Currently there is nocapability to enable mass spectrometric characterization of dynamic,living systems without destroying or negatively altering the entiresystem. Methods to provide mass spectrometric in situ chemicalinformation living systems are of interest to the biochemical,pharmaceutical, and medical research communities.

SUMMARY OF THE INVENTION

A method for sampling a sample liquid includes the step of providing thesample liquid through a sample fluid conduit. The sample fluid conduitincludes a membrane including pores. The membrane prevents the passageof the sample liquid through the pores at a first pressure of the sampleliquid in the sample fluid conduit.

A surface sampling capture probe with a distal end is provided. Thecapture probe includes a solvent supply conduit with an open end and asolvent exhaust conduit with an open end. A solvent composition isflowed at the distal end of the capture probe from the open end of thesolvent supply conduit to the open end of the exhaust conduit andestablishes a liquid junction with the membrane and establishes a secondpressure within the liquid junction at the membrane. The second pressurecan be lower than the first pressure. Sample liquid is drawn through thepores of the membrane by the second pressure at the liquid junctionwherein the extracted sample liquid is combined with the flowing solventcomposition of the capture probe and flows into the open end of theexhaust conduit of the capture probe. The method can further include thestep of conducting mass spectrometry on the extracted sample liquid. Themethod can further include the step of collecting the solventcomposition and sample liquid from the exhaust conduit of the captureprobe in a storage container.

The surface tension of the solvent composition when in a liquid junctionwith the membrane having a junction fluid diameter can prevent expansionof the junction fluid diameter beyond two times the diameter of thedistal end of the capture probe. The surface tension of the solventcomposition does not permit the solvent to flow through the pores of themembrane at the second pressure.

The solvent composition can include a component that is a solvent for ananalyte of interest in the sample liquid. The solvent composition caninclude a solvent liquid and a high surface tension component having asurface tension higher than that of the solvent liquid. The solventcomposition can include an acid or base component for ionizing ananalyte of interest in the sample fluid. The solvent composition caninclude a reactant for an analyte of interest in the sample liquid toproduce an analyte reaction product. The analyte reaction productprovides increased sensitivity for mass spectrometry relative to theunreacted analyte of interest.

A system for sampling a liquid can include a sample fluid conduit. Thesample fluid conduit can include a membrane comprising pores. Themembrane prevents the passage of the sample liquid through the pores ata first pressure of the sample liquid in the sample fluid conduit. Asurface sampling probe with a distal end can be provided. The captureprobe can include a solvent supply conduit with an open end and asolvent exhaust conduit with an open end. A solvent composition flowingat the distal end of the capture probe from the open end of the solventsupply conduit to the open end of the exhaust conduit can establish aliquid junction with the membrane and can establish a second pressurewithin the liquid junction at the membrane. The second pressure can belower than the first pressure. Sample liquid will be drawn through thepores of the membrane by the second pressure at the liquid junction andthe extracted sample liquid will combine with the flowing solventcomposition of the capture probe and flow into the open end of theexhaust conduit of the capture probe. The system can further include achemical analysis device. The chemical analysis device can include amass spectrometer.

The capture probe can include an outer probe housing having a coaxialinner solvent exhaust conduit, and an annular solvent supply conduitbetween the solvent exhaust conduit and the outer prove housing. Thesystem can further include a storage container for collecting thesolvent composition and sample liquid from the exhaust conduit of thecapture probe. The sample fluid conduit can be provided in a samplefluid conduit housing.

A method of performing chemical analysis on a sample liquid can includethe step of providing the sample liquid through a sample fluid conduit.The sample fluid conduit can include a membrane comprising pores. Themembrane prevents the passage of the sample liquid through the pores ata first pressure of the sample liquid in the sample fluid conduit. Asurface sampling capture probe with a distal end is provided. Thecapture probe can include a solvent supply conduit with an open end anda solvent exhaust conduit with an open end. The method includes flowinga solvent composition at the distal end of the capture probe from theopen end of the solvent supply conduit to the open end of the exhaustconduit and establishing a liquid junction with the membrane andestablishing a second pressure within the liquid junction at themembrane. The second pressure can be lower than the first pressure.Sample liquid is drawn through the pores of the membrane by the secondpressure at the liquid junction, and the extracted sample liquid iscombined with the flowing solvent composition of the capture probe andflows into the open end of the exhaust conduit of the capture probe. Theextracted sample liquid is directed to a chemical analysis device andperforming chemical analysis on the extracted sample liquid. Thechemical analysis device can be a mass spectrometer and the chemicalanalysis can be mass spectrometry.

A method for sampling a sample liquid can include the step of providingthe sample liquid in a sample fluid conduit. The sample fluid conduitcan include a membrane comprising pores. The membrane prevents thepassage of the sample liquid through the pores at a first pressure ofthe sample liquid in the sample fluid conduit. Solvent composition isflowed from the open end of a solvent supply conduit to contact themembrane. A second pressure is established within the solvent in contactwith the membrane through a solvent exhaust conduit. The second pressureis lower than the first pressure. Sample liquid is drawn through thepores of the membrane by the pressure differential of the first pressureand the second pressure, wherein the extracted sample liquid is combinedwith the flowing solvent composition and flows into the open end of asolvent exhaust conduit.

The solvent supply conduit and the solvent exhaust conduit can be thesame conduit, for example the supply and exhaust of solvent can beprovided by varying the pressure within a single tube having an openend. A pipette is an example. The solvent can form a liquidmicrojunction between the tube and the membrane. The solvent can bedeposited on the membrane by the solvent supply conduit, then the supplyof solvent can be stopped and the solvent on the membrane allowed tostand for a period of time, and then the second pressure can be appliedto the solvent in contact with the membrane by the solvent exhaustconduit.

BRIEF DESCRIPTION OF THE DRAWINGS

There are shown in the drawings embodiments that are presently preferredit being understood that the invention is not limited to thearrangements and instrumentalities shown, wherein:

FIG. 1 is a schematic cross-sectional diagram of a system for performingmass spectrometry analysis on a flowing liquid sample source.

FIG. 2 is a schematic cross sectional diagram of a microfluidic devicefor performing chemical analysis of a flowing liquid sample source.

FIG. 3 is an enlarged schematic cross sectional diagram of area

FIG. 3 in FIG. 1.

FIG. 4 is a an enlarged schematic diagram of area FIG. 3 in FIG. 1, andin an alternative mode of operation.

FIG. 5 is an extracted ion chronogram of m/z 195.1 (black) and m/z 198.1(grey) corresponding to caffeine and d₃-caffeine, respectively.

FIG. 6 is a plot of fluid extraction efficiency through the porousmembrane (vol %) vs. capture probe-to-surface distance for flow cellflow rate=10 μL/min, capture probe flow rate=151 μL/min, and membranepore size=0.4 μm.

FIG. 7 is a plot of fluid extraction efficiency through the porousmembrane (vol %) vs. capture probe flow rate (μL/min) for flow cell flowrate=10 μL/min, capture probe-to-surface distance=20 μm, and membranepore size=0.4 μm.

FIG. 8 is a plot of fluid extraction efficiency through the porousmembrane (vol %) vs. membrane pore size (μm) for flow cell flow rate=10μL/min, capture probe flow rate=150 μL/min, and capture probe-to-surfacedistance=20 μm.

FIG. 9 is a plot of fluid extraction efficiency (vol %) vs. flow cellflow rate (μL/min) for capture probe flow rate=150 μL/min, captureprobe-to-surface distance=20 μm, and membrane pore size=0.4 μm.

FIG. 10 is an extracted ion chronogram showing intensity (a.u.) vs. time(min) for m/z 195.1 (black) and m/z 198.1 (grey) corresponding tocaffeine and d₃-caffeine signals.

FIG. 11 is an optical image of a microfluidic flow cell with a Y-shapedchannel, with the dashes representing the area selected for massspectrometry imaging.

FIG. 12 is an MS image of the dashed area in FIG. 12 showing propranolol(m/z 260.1, light grey) and caffeine (m/z 195.1, grey) in the flow cell.The scale bar represents 2 mm.

FIG. 13 A-H are schematic depictions of a method of sampling a sampleliquid according to an alternative embodiment.

DETAILED DESCRIPTION OF THE INVENTION

A method for sampling a sample liquid includes the step of providing thesample liquid through a sample fluid conduit. The sample fluid conduitcomprises a membrane comprising pores. The membrane prevents the passageof the sample liquid through the pores at a first pressure of the sampleliquid in the sample fluid conduit. A surface sampling capture probe isprovided having a distal end. The capture probe includes a solventsupply conduit with an open end and a solvent exhaust conduit with anopen end. Solvent composition flows at the distal end of the captureprobe from the open end of the solvent supply conduit to the open end ofthe exhaust conduit, and establishes a liquid junction with themembrane. A second pressure is established within the liquid junction atthe membrane. The second pressure is lower than the first pressure,thereby drawing sample liquid through the pores of the membrane by thesecond pressure at the liquid junction. The extracted sample liquid iscombined with the flowing solvent composition of the capture probe andflows into the open end of the exhaust conduit of the capture probe.

A system for sampling a liquid includes a sample fluid conduit includinga membrane having pores. The membrane barrier prevents the passage ofthe sample liquid through the pores at a first pressure of the sampleliquid in the sample fluid conduit. A surface sampling capture probe hasa distal end. The capture probe includes a solvent supply conduit withan open end and a solvent exhaust conduit with an open end. A solventcomposition flowing at the distal end of the capture probe from the openend of the solvent supply conduit to the open end of the exhaust conduitestablishes a liquid junction with the membrane and establishes a secondpressure within the liquid junction at the membrane. The second pressureis lower than the first pressure. Sample liquid will be drawn throughthe pores of the membrane by the second pressure at the liquid junctionand the extracted sample liquid combines with the flowing solventcomposition of the capture probe and flow into the open end of theexhaust conduit of the capture probe. A method for sampling a liquid andfor performing chemical analysis on a liquid are also disclosed.

The method can further include the step of conducting mass spectrometryon the extracted sample liquid. The system can also be used to withdrawsample from a flowing sample liquid for other chemical analysis methodsand devices. The method can include the step of collecting the solventcomposition and sample liquid from the exhaust conduit of the captureprobe in a storage container for later analysis by any suitable chemicalanalysis method or device. The system can include a storage containerfor collecting the solvent composition and sample liquid from theexhaust conduit of the capture probe. The storage container can havediffering designs and sizes.

The method can utilize a number of different solvent compositions. Thesolvent composition when in a liquid junction with the membrane has ajunction fluid diameter, and the surface tension of the solventcomposition and selection of the membrane material should preventexpansion of the junction fluid diameter beyond two times the diameterof the distal end of the capture probe. The surface tension of thesolvent composition should also not permit the solvent composition toflow through the pores of the membrane at the second pressure. Suitablesolvents include methanol or acetonitrile mixed with water, formic acid,ammonium acetate, ammonium hydroxide, acetic acid, ammonium formate, orisopropyl alcohol.

The solvent composition can include a component that is a solvent for ananalyte of interest in the sample liquid, for example, methanol oracetonitrile. The solvent composition can include a solvent liquid and ahigh surface tension component having a surface tension higher than thatof the solvent liquid for example, water or isopropyl alcohol. Thesolvent composition can include an acid or base component for ionizingan analyte of interest in the sample fluid for example, formic acid,ammonium acetate, ammonium hydroxide, acetic acid or ammonium formate.The solvent composition can include a reactant for an analyte ofinterest in the sample liquid to produce an analyte reaction productwhich analyte reaction product provides increased sensitivity for massspectrometry relative to the unreacted analyte of interest for example,the molecular tagging of cysteines with benzoquinones.

The system can further include a chemical analysis device. The chemicalanalysis device can include a mass spectrometer. Other chemical analysismethods and devices that are suitable for use with the invention includespectroscopic detection (fluorescence, Raman, etc.), conductivitymeasurement, or pH detection.

The capture probe can have different designs. In one aspect, the captureprobe comprises an outer probe housing having a coaxial inner solventexhaust conduit, and an annular solvent supply conduit between thesolvent exhaust conduit and the outer prove housing.

The surface tension of the solvent composition does not permit thesolvent composition to flow through the pores of the membrane at thesecond pressure. It is possible to operate the capture probe at a thirdpressure, less than the pressure of the sample fluid but not low enoughto draw significant amounts of the sample fluid through the pores.

The construction of the membrane can vary. The membrane can be made frompolycarbonate (PCT), polyester (PET), polytetrafluoroethylene (PETE),polydimethylsiloxane (PDMS), polyether ether ketone (PEEK), nylon,silver or glass Fiber. The membrane pores can have differing dimensions,for example from 0.01 to 10 μm. The thickness of the membrane can vary,for example from 0.1 to 1000 μm. The membrane can have track-etched ortortuous pores and can be coated with polymer films and can have 1 to10⁸ pores/cm².

The sample fluid conduit can be provided in a sample fluid conduithousing. The housing can have differing designs and sizes. The samplefluid conduit can be provided in a microfluidic device. The fluid doesnot have to flow constantly, for example, fluid can be flowed into thecell, halted and then sampled.

There is shown in FIGS. 1-4 a system 10 for porous membrane enabled massspectrometry characterization of microfluidic devices. The system 10includes a capture probe 14 and a suction nebulizer 18. The captureprobe 14 can include an outer tube 22 with distal open end 23 and aninner tube 26 with distal open end 27 arranged in substantially coaxialarrangement. Other capture probe designs are possible.

The capture probe 14 receives solvent from a suitable solvent source 30.Solvent received from source 30 travels through the capture probe 14 inannular solvent supply conduit 38 formed between the outer tube 22 andthe inner tube 26. The inner tube 26 defines an exhaust conduit 42 andhas a distal end 27. The suction nebulizer 18 applies suction as by asuitable pump or in the embodiment shown a supply of nebulizer gas 46which is introduced into nebulizer passageway 48 through inlet 50 andflows past open end 54 of exhaust passageway 44. The flow of nebulizergas contacts solvent at the open end 54 to create a nebulizer spray 56of the sample solvent into MS inlet 58. The flow of nebulizer gascreates a low pressure at the open end 54 and thereby within the exhaustconduit 44 and within the exhaust conduit 42 of the capture probe 14 italso creates a low-pressure zone in the exhaust conduit 42 within theinner tube 26. Control of the suction nebulizer 18 can be affected tocontrol the flow of solvent through the exhaust conduit 26. Control ofthe solvent source can be affected through a suitable valve 32 throughan inlet opening 34. The supply of solvent and the exhaust of solventcan be controlled by a suitable processor. The balance of solvent flowsin the solvent supply conduit 38 and the solvent exhaust conduit 42creates a liquid microjunction 106.

A microfluidic device 60 includes a wall 64 and a porous membrane 68defining a flow channel 76. The porous membrane 68 includes pores 72. Asample solution 75 containing analyte 77 is provided in the flow channel76 at pressure P₁. The sample solution can enter the flow channel 76 atany suitable fluid inlet 80 and exit at a suitable fluid exit 84. Thesample fluid can flow in the flow channel 76 as indicated by arrow 90.The size of the pores 72 and the surface tension of the sample fluid issuch that the sample fluid may enter the pores 72 as shown by meniscus78, but does not pass through the pores 72. The sample solution 76 iscontained by the microfluidic device 60 at pressure P₁ when the pressureoutside the microfluidic device 60 is that pressure P₀. P₀ can be anyoutside pressure, such as atmospheric pressure.

In operation, as shown in FIG. 3, the capture probe 14 is brought intoproximity to the microfluidic device 60 and solvent is caused to flowthrough the annular solvent supply conduit 38 solution as shown byarrows 98. The section nebulizer 18 is operated to withdraw solventsolution through the exhaust conduit 42 as indicated by arrow 102. Thiswill cause the formation of a liquid microjunction 106 between distalend 23 of the capture probe 14 and the porous membrane 68. The balancebetween solvent supply and withdrawal can be controlled to create apressure P₂ within the liquid microjunction 106. P₂ is less than P₀ andless than P₁. At P₂ the pressure differential between P₂ and P₁ issufficient to overcome the surface tension of the solvent solution 75within the pores 72 such that some sample fluid 110 will be drawnthrough the pores 72 and into the exhaust conduit 42.

The pressure within the liquid microjunction can be controlled bycontrol of the solvent supply rate and the solvent exhaust rate. FIG. 4illustrates the condition where the supply and exhaust solvent flows arecontrolled to provide a liquid microjunction 114 with a pressure P₃within the microjunction 114. The pressure P₃ is less that P₁, butgreater than P₂. The pressure differential between P₁ and P₃ isinsufficient to draw sample solution 75 through the pores 72, and isinsufficient to drive solvent from the capture probe 14 into the flowchannel 76. P₃ should not be so great that solvent is forced through thepores 72 and into the flow channel 76 microfluidic device 60 as thiscould contaminate or dilute the sample solution 75 in the flow channel76. The system can be controlled on demand to change the solvent flowrates to a pressure, for example P₂, that will withdraw sample fluidfrom the membrane 76.

The invention provides for the on-line/in situ chemical analysis ofmicrofluidic flow cells through the combination of a microfluidic devicewith a porous polymer membrane wall and sampling with liquidmicrojunction-surface sampling capture probe mass spectrometry (MS).This enables online chemical characterization of constituents within thedevice by MS. This combination provides a system and method tocontinuously sample and chemically characterize small volumes of liquiddirectly from a microfluidic device at any point along a 2D surface innear real-time and without greatly negatively altering the state of themicrofluidic system. The invention can also be used for the chemicalanalysis of living systems in microfluidic devices continuously withoutdestruction or modification to the biological system.

The microfluidic device is fabricated with a porous membrane surface onone side of the device, or on several sides. The capture probe MSsampling device can form a liquid microjunction between the membranesurface which extracts nL-to-uL volumes from the microfluidic device,through the membrane and into the capture probe. The liquid extracts canthen be chemically characterized by mass spectrometry, or anotherchemical analysis technology. The porous membrane of the microfluidicdevice also allows selective liquid extraction at any point along themembrane surface. The use of a porous membrane microfluidic device in aconfiguration that allows for chemical analysis at any point on thesurface of the device, without destroying the function and lifetime ofthe device.

Chemical analysis of dynamic fluid systems often precludes the use ofmass spectrometry as it is destructive. The invention provides a way tomeasure the chemistry within a microfluidic flow cell at any point alonga 2D surface using mass spectrometry. Uses include the on-line measureof chemical dynamics within microfluidic devices with mass spectrometricdetection. Chemical mapping of over the entire surface of a microfluidicdevice is possible to show chemical differences occurring in differentregions of the device. The invention permits repeated chemical measureat single or multiple points anywhere on the microfluidic deviceproviding temporally resolved chemical information. Mass spectrometricchemical detection within microfluidic devices can be performed withoutdismantling the device or negatively influencing its function.

The capture probe that was used also included a vacuum suctioncapillary. The capture probe was made using three capillaries co-axiallyaligned. The outer capillary was stainless steel (˜1.067 mm-i.d.×˜0.686mm-o.d.×3.5 cm long) connected to a PEEK T-manifold (IDEX Health &Science LLC, Oak Harbor, Wash., USA). One of the ports connected to thesampling end of the capture probe and the other connected to housevacuum to provide suction through this tube. This served to prevent anypooling of solvent on the sample surface when solvent delivery exceededaspiration at the initialization of the capture probe device, but it didnot influence normal capture probe operation. The middle capillary wasstainless steel (˜0.406 mm-i.d.×0.508 mm-o.d.×7.0 cm long) connected tothe mass spectrometer ion source electrical ground. A stainless-steelcapillary (0.178 μm-i.d.×0.330 μm-o.d.×˜30 cm long) was used as theinner tube and acted as the nebulizing capillary of an atmosphericpressure chemical ionization (APCI) ion source of a Q-Exactive HForbitrap mass spectrometer (ThermoFisher Scientific, Waltham, Mass.,USA). The middle and inner capillaries were secured to another PEEKT-manifold (IDEX Health & Science LLC, Oak Harbor, Wash., USA). Solventwas delivered by a high-performance liquid chromatography (HPLC) pump(model 1100, Agilent Technologies, Santa Clara, Calif., USA) into theannulus region of the capture probe. Nebulizing gas (nitrogen) in theion source was used to control the solvent aspiration rate through theprobe. This was set to slightly exceed the solvent delivery rate to thecapture probe from the HPLC.

A stable liquid vortex was maintained at the surface of the probe. Uponcontact of the capture probe with a surface, a liquid microjunction wasformed between the surface and the capture probe. The capture probe wasconnected to a XYZ stage (MS-2000, Applied Scientific Instrumentation,Eugene, Oreg., USA) which was used to control the movement of the probe.Control of the stage was done using custom control software developed inhouse using Delphi 3 computer language (Borland Software Corp., ScottsValley, Calif.). Mass spectra were acquired continuously using a sheathgas flow=80, auxiliary gas flow=40, capillary temperature=250° C.,source temperature=400° C., and APCI current=5 μA. Specific scansettings were optimized for each experiment. Caffeine and d₃-caffeinewere monitored simultaneously using all ion fragmentation (AIF) scans ofm/z 190-200 with a scan window of m/z 100-200 (normalized collisionenergy (NCE)=50 eV, resolution=45,000, automatic gain control (AGC)=1e6,injection time=100 ms). A common fragment ion to caffeine andd₃-caffeine (m/z 138) was used to corroborate experiments.

The invention permits the efficient extraction of small amounts of fluidfrom a microfluidic flow cell with a porous membrane conducted in situwithout significantly affecting its operation. To validate the abilityto extract fluid through the porous membrane wall, a single-channel (500μm wide and 160 μm deep) microfluidic flow cell was designed. Thechannel was sealed using a 0.4 μm pore size track etched, hydrophobicPETE membrane. The flow cell was fed with a constant 10 μL/min flow of2.55 μM caffeine in water. Based on the pore size of the membrane andthe surface tension of water, the 2.55 μM caffeine solution flowedthrough the channel rather than out of the porous membrane.

The solvent of the capture probe was optimized to achieve efficientanalyte extraction without negatively impacting the flow cell.Typically, solvents such as acetonitrile and methanol are used incapture probe operation, but these solvents have sufficiently lowsurface tension that they can wet the entire membrane surface uponcontact with the capture probe. A solvent composition of 75/25/0.1ACN/H₂O/FA (v/v/v) was found to enable liquid microjunction formationwithout wetting of the PETE membrane beyond the area of the captureprobe. To demonstrate the continuous fluid extraction through the porousmembrane, the capture probe was positioned ˜20-40 μm above the membranesurface in order to form a liquid microjunction between the membranesurface and the capture probe. Liquid extraction through the PETEmembrane was monitored by mass spectrometric detection of caffeine thatwas present in the solvent flowing through the flow cell. To quantitatethe amount of caffeine detected, d₃-caffeine was added to the captureprobe extraction solvent to act as an internal standard. The captureprobe extraction solvent was made up by mixing 75/25/0.1 ACN/H₂O/FA at150 μL/min with 44 μM d₃-caffeine in water (final concentration of 0.29μM) at 1 μL/min.

FIG. 5 shows extracted ion chronograms of m/z 195.1 (black) and m/z198.1 (grey) corresponding to caffeine and d₃-caffeine, respectively.FIG. 5 shows continuous fluid extraction through the PETE membrane bythe capture probe. A liquid microjunction was formed at t=1 min, whichimmediately resulted in a mass spectrometric signal of caffeine (˜6 selution time). The capture probe remained in this position for 14 minbefore it was lifted away from the surface, breaking the liquidmicrojunction. This is reflected by the sharp decrease in caffeinesignal at t=15 min. Continuous extraction of caffeine was observed overthe 14 min time period where the liquid microjunction was in place.Hence, this data shows continuous extraction of small volumes of liquidfrom the flow cell through the porous membrane and into the captureprobe. D₃-caffeine signal (grey line) was approximately constant overthe duration indicating no matrix effects were observed and that no lossof capture probe solvent, wicking of solvent into the flow cell, wasoccurring.

One concern of using capture probe to extract liquid through the porousmembrane wall is the transport of capture probe solvent into the flowcell itself. If this occurs, then the act of sampling by a liquidmicrojunction may negatively alter or dilute the chemistry occurring inthe flow cell. Additionally, for biological systems, exposure torelatively harsh extraction solvents may negatively impact the system.To determine if and to what extent capture probe solvent enters the flowcell through the porous membrane, 100% water without any analyte wasinfused through the flow cell and outflows were collected under varyingflow rates (5-15 μL/min) with and without liquid microjunction formationover the PETE membrane. Since 0.29 μM d₃-caffeine is present only in thecapture probe solvent, any d₃-caffeine signal in collected outflows isfrom transfer of capture probe solvent into the flow cell. D₃-caffeinesignals were statistically the same (99% confidence) without captureprobe sampling and with capture probe sampling using 5, 10 and 15 μL/minwater flow rate in the flow cell. This result indicates that captureprobe solvent had not entered the flow cell. Based on the limit ofdetection of the mass spectrometer in this configuration for d₃-caffeine(˜1 nM), this data shows that <0.4% of capture probe solvent had beenintroduced into the flow cell. This data is also corroborated by theconstant d₃-caffeine signals observed in FIG. 5. If capture probesolvent entered the flow cell there would be reduced d₃-caffeine signal,which was not observed.

Control of the extent of liquid extraction can be tuned for eachapplication to minimally perturb the system within the device. Liquidextraction through the porous membrane is likely influenced by severalfactors including porous membrane pore size, liquid flow rate in theflow cell and capture probe sampling parameters, such as extractionsolvent, flow rate, capture probe-to-surface distance, and capture probecapillary dimensions. Briefly, the inner capillary retraction length andcapture probe-to-surface distance were found to be critical factors thatgovern surface sampling. Here, inner capillary retraction lengths werefixed at ˜500 μm, but the distance of the capture probe from themembrane surface could vary as the capture probe is moved.

The effect of capture probe-to-surface distance, the thickness of theliquid microjunction, on fluid extraction efficiency is shown in FIG. 6.Extraction efficiencies were determined by measuring the mass of flowcell outflows and by quantitation of caffeine by capture probe-MS. Fluidextraction efficiency through the porous membrane as a function ofcapture probe-to-surface distance is shown in FIG. 6 for flow cell flowrate=10 μL/min, capture probe flow rate=151 μL/min, and membrane poresize=0.4 μm. Fluid extraction efficiency is the fraction of flow cellflow rate pulled into the capture probe, and was determined directly byweighing the mass of flow cell outflows with and without capture probesampling and by quantitation of a known concentration of analyte presentin the flow cell by capture probe-MS. In the latter method the amountmass spectrometric signal of caffeine was quantitated and used todetermine how much flow cell fluid became integrated into the captureprobe. In the flow cell 10 μL/min of 2.55 μM caffeine in water wascontinuously flowed and a membrane with a pore size of 0.4 μm was usedto seal the device. The capture probe used a flow rate of 151 μL/min0.29 μM d₃-caffeine in 75/25/0.1 (v/v/v) ACN/H₂O/FA. Flow cell outflowsfor each capture probe-to-surface distance were collected into 1 mLEppendorf tubes for 3 min and weighed. Blanks (no capture probesampling) were used as a reference for normal flow cell outflow masses.

In the same experiment the concentration of caffeine sampled by captureprobe was calculated by measure of caffeine and d₃-caffeine by captureprobe-MS. Caffeine signal was normalized to the internal standard andtransformed into a quantitative concentration using an externallymeasured calibration curve. Three replicate experiments were conductedfor each condition. Given the flow rate through the flow cell is known,extracted flow rates were converted relative to the fraction of flowcell flow rate extracted (FIG. 6) to facilitate comparisons betweenconditions. FIG. 6 shows that the capture probe-to-surface distanceinversely correlates with fluid extraction efficiency, with ˜30% of flowcell fluid sampled by the capture probe when position directly over themembrane surface (capture probe-to-surface distance=0 μm). Above 60 μmthe liquid microjunction could no longer be reliably maintained.

Using the MS quantitation methodology outlined for the experiments inFIG. 6, the capture probe flow rate was varied from 75-175 μL/min using0.29 μM d₃-caffeine in 75/25/0.1 (v/v/v) ACN/H₂O/FA while keeping theflow cell flow rate, capture probe-to-surface distance and MS ion sourceaspiration conditions constant. FIG. 7 shows a capture probe flow ratewith flow cell flow rate=10 μL/min, capture probe-to-surface distance=20μm, and membrane pore size=0.4 μm. As the capture probe solvent flowrate increased the fluid extraction efficiency decreased. Above 175μL/min the capture probe solvent flow rate becomes greater than theself-aspiration rate of the MS ion source, resulting in the captureprobe overflowing with solvent and no fluid being extracted from theflow cell. As the capture probe solvent flow rate is lowered below 175μL/min, there was greater extraction efficiency through the porousmembrane. An improvement of liquid extraction by ˜10 fold was achievedby decreasing capture probe flow rate from 175 to 75 μL/min (3% to 20%,respectively). Increased extraction efficiency with decreasing captureprobe flow rate is explained by considering the effective negativepressure exerted by the capture probe. A flow rate of 151 μL/min wasused for the remainder of these experiments.

Fluid extraction efficiency was determined using membrane pore sizes of0.1, 0.2, 0.4 and 5 μm in diameter (FIG. 8) while keeping capture probeflow rate, capture probe-to-surface distance and MS ion sourceaspiration conditions constant. FIG. 8 shows data for flow cell flowrate=10 μL/min, capture probe flow rate=150 μL/min, and captureprobe-to-surface distance=20 μm. At 5 μm pore diameter 92% of the flowcell fluid is extracted by the capture probe, while for the 0.1 μm poresize membrane this dropped to 1% (0.1 μL/min). Smaller pore sizerequires greater vacuum pressure to extract the same amount of liquidthrough the membrane.

FIG. 9 shows the extracted flow rate dependence on the fluid flow ratewithin the flow cell. The increasing flow cell flow rate is inverselyrelated to the amount of extracted flow. Extraction efficiencies weredetermined by measuring the mass of flow cell outflows and byquantitation of caffeine by capture probe-MS.

To demonstrate reproducible and repeated sampling across the porousmembrane surface of the channel the capture probe was continuouslyrastered forward and backward 27 times in 15 min across the samelocation of the channel at a rate of 0.1 mm/s (FIG. 10). Effectively,these scans demonstrate periodic monitoring of the fluid channel overseveral minutes. A 2.55 μM caffeine solution was continuously flowedthrough the flow cell at a rate of 10 μL/min. Transects across thechannel result in a Gaussian-like signal profile that remainedconsistent for all 27 replicate scans.

The use of a porous membrane enables sampling and chemical imaging ofdynamic microfluidic devices. As a proof-of-concept a microfluidicdevice with a two-input Y-tee channel was fabricated and sealed using a0.4 μm porous PETE membrane (FIG. 11). FIG. 11 shows optical image ofthe microfluidic flow cell with a Y-shaped channel. The dashed area wasselected for MS imaging in FIG. 12. Input channels were 0.75 mm wide and0.165 mm deep, and the combined channel was 1 mm wide and 0.165 mm deep.To validate the imaging resolution and accuracy of the chemical imagingcapability, input channel #1 was fed with 15 μL/min of 2.55 μMpropranolol (a.q.) and input channel #2 was fed with 15 μL/min of 2.55μM caffeine (a.q.). The two inputs and the central channel were thenimaged using the capture probe-MS system (FIG. 12). FIG. 12 showsextracted ion chronograms of m/z 195.1 (black) and m/z 260.1 (lightgrey) corresponding to caffeine and proporanolol, respectively. To imagethe surface the capture probe was rastered across the flow cell at 0.25mm/s, with 0.25 mm spacing between lanes for 41 lanes. The imagecomprised a 10×10 mm surface area (41×41 px image). The resultingcapture probe MS image aligned well with the flow cell design. Since themembrane surface is relatively flat, a constant capture probe-to-surfacedistance could be maintained throughout the imaging experiment. Asexpected with this type of device, the two solutions fed into theindividual input channels remain separate even within the central(combined) channel. Since input flow rates are relatively high, thevelocity of fluid in the mixed channel is ˜3 mm/s (30 μL/min, 1×0.165 mmrectangular channel). Thus, there is very little time in the imaged areafor the two components to mix (<2 sec). This is observed clearly in theMS image where the two components can be seen separated in the centralchannel. These images demonstrate the ability of capture probe toidentify chemically heterogenous regions within a dynamic systemanywhere along the porous membrane surface. Note, the same device can beimaged repeatedly to monitor for changes over time.

There is shown in FIGS. 13A-13H an embodiment of the invention in whicha single conduit 124 serves as one or both of the solvent supply conduitand the solvent/sample exhaust conduit. Positive pressure is applied tosolvent within the conduit 124 to cause solvent to be expelled from theopen end 126 of the conduit 124 and form a liquid microjunction with themembrane 122. This is followed by subsequent withdrawal of a sample fromthe surface such as by applying a reduced or negative pressure withinthe conduit 124. This will create a low pressure within the liquidmicrojunction that is less than the pressure of the sample liquid on theother side of the membrane 122. For example, in FIG. 13A, the collectionprobe 124 is positioned above the surface spot to be analyzed. In FIGS.13B through 13E, the liquid solvent conducted upon the surface begins tobuild up upon the surface. In FIGS. 13F through 13H, the pressure withinthe conduit 124 is reduced so that an analyte-rich solution is drawnthrough the membrane 122, and into the conduit 124 or a separate exhaustconduit.

It can be seen from the view of FIGS. 13B through 13E, the area or spotover which the sample is covered by the liquid solution increases insize (i.e. diameter) as the liquid solution accumulates upon thesurface. Such an occurrence can be advantageous in that the solutionwhich is subsequently withdrawn from the surface for analysis containssample amounts from the relatively broad area covered by the liquidagent.

The invention as shown in the drawings and described in detail hereindisclose arrangements of elements of particular construction andconfiguration for illustrating preferred embodiments of structure andmethod of operation of the present invention. It is to be understoodhowever, that elements of different construction and configuration andother arrangements thereof, other than those illustrated and describedmay be employed in accordance with the spirit of the invention, and suchchanges, alternations and modifications as would occur to those skilledin the art are considered to be within the scope of this invention asbroadly defined in the appended claims. In addition, it is to beunderstood that the phraseology and terminology employed herein are forthe purpose of description and should not be regarded as limiting.

We claim:
 1. A method for sampling a sample liquid, comprising the stepsof: providing the sample liquid through a sample fluid conduit, thesample fluid conduit comprising a membrane comprising pores, themembrane preventing the passage of the sample liquid through the poresat a first pressure of the sample liquid in the sample fluid conduit;providing a surface sampling capture probe with a distal end, thecapture probe comprising a solvent supply conduit with an open end and asolvent exhaust conduit with an open end; flowing solvent composition atthe distal end of the capture probe from the open end of the solventsupply conduit to the open end of the exhaust conduit and establishing aliquid junction with the membrane and establishing a second pressurewithin the liquid junction at the membrane, the second pressure beinglower than the first pressure; drawing sample liquid through the poresof the membrane by the second pressure at the liquid junction whereinthe extracted sample liquid is combined with the flowing solventcomposition of the capture probe and flows into the open end of theexhaust conduit of the capture probe.
 2. The method of claim 1, furthercomprising the step of conducting mass spectrometry on the extractedsample liquid.
 3. The method of claim 1, wherein the surface tension ofthe solvent composition when in a liquid junction with the membranehaving a junction fluid diameter prevents expansion of the junctionfluid diameter beyond two times the diameter of the distal end of thecapture probe.
 4. The method of claim 1, wherein the surface tension ofthe solvent composition does not permit the solvent to flow through thepores of the membrane at the second pressure.
 5. The method of claim 1,further comprising the step of collecting the solvent composition andsample liquid from the exhaust conduit of the capture probe in a storagecontainer.
 6. The method of claim 1, wherein the solvent compositioncomprises a component that is a solvent for an analyte of interest inthe sample liquid.
 7. The method of claim 1, wherein the solventcomposition comprises a solvent liquid and a high surface tensioncomponent having a surface tension higher than that of the solventliquid.
 8. The method of claim 1, wherein the solvent compositioncomprises an acid or base component for ionizing an analyte of interestin the sample fluid.
 9. The method of claim 1, wherein the solventcomposition comprises a reactant for an analyte of interest in thesample liquid to produce an analyte reaction product which analytereaction product provides increased sensitivity for mass spectrometryrelative to the unreacted analyte of interest.
 10. A system for samplinga liquid, comprising: a sample fluid conduit, the sample fluid conduitcomprising a membrane comprising pores, the membrane preventing thepassage of the sample liquid through the pores at a first pressure ofthe flowing sample liquid in the sample fluid conduit; a surfacesampling probe with a distal end, the capture probe comprising a solventsupply conduit with an open end and a solvent exhaust conduit with anopen end, wherein a solvent composition flowing at the distal end of thecapture probe from the open end of the solvent supply conduit to theopen end of the exhaust conduit establishes a liquid junction with themembrane and establishes a second pressure within the liquid junction atthe membrane, the second pressure being lower than the first pressure;wherein sample liquid will be drawn through the pores of the membrane bythe second pressure at the liquid junction and the extracted sampleliquid will combine with the flowing solvent composition of the captureprobe and flow into the open end of the exhaust conduit of the captureprobe.
 11. The system of claim 10, further comprising a chemicalanalysis device.
 12. The system of claim 11, wherein the chemicalanalysis device comprises a mass spectrometer.
 13. The system of claim10, wherein the surface tension of the solvent composition when in aliquid junction with the membrane having a junction fluid diameterprevents expansion of the junction fluid diameter beyond two times thediameter of the distal end of the capture probe.
 14. The system of claim10, wherein the capture probe comprises an outer probe housing having acoaxial inner solvent exhaust conduit, and an annular solvent supplyconduit between the solvent exhaust conduit and the outer prove housing.15. The system of claim 10, wherein the surface tension of the solventcomposition does not permit the solvent composition to flow through thepores of the membrane at the second pressure.
 16. The system of claim10, further comprising a storage container for collecting the solventcomposition and sample liquid from the exhaust conduit of the captureprobe.
 17. The system of claim 10, wherein the solvent compositioncomprises a component that is a solvent for an analyte of interest inthe sample liquid.
 18. The system of claim 10, wherein the solventcomposition comprises a high surface tension component.
 19. The systemof claim 10, wherein the solvent composition comprises an acid or basecomponent for ionizing an analyte of interest in the sample fluid. 20.The system of claim 10, wherein the sample fluid conduit is provided ina sample fluid conduit housing.
 21. The system of claim 10, wherein thesolvent composition comprises a reactant for an analyte of interest inthe sample liquid to produce an analyte reaction product which analytereaction product provides increased sensitivity for mass spectrometryrelative to the unreacted analyte of interest.
 22. A method for samplinga sample liquid, comprising the steps of: providing the sample liquidthrough a sample fluid conduit, the sample fluid conduit comprising amembrane comprising pores, the membrane preventing the passage of thesample liquid through the pores at a first pressure of the sample liquidin the sample fluid conduit; providing a surface sampling capture probewith a distal end, the capture probe comprising a solvent supply conduitwith an open end and a solvent exhaust conduit with an open end; flowinga solvent composition at the distal end of the capture probe from theopen end of the solvent supply conduit to the open end of the exhaustconduit and establishing a liquid junction with the membrane andestablishing a second pressure within the liquid junction at themembrane, the second pressure being lower than the first pressure;wherein sample liquid is drawn through the pores of the membrane by thesecond pressure at the liquid junction and wherein the extracted sampleliquid is combined with the flowing solvent composition of the captureprobe and flows into the open end of the exhaust conduit of the captureprobe; directing the extracted sample liquid to a chemical analysisdevice and performing chemical analysis on the extracted sample liquid.23. The method of claim 22, wherein the chemical analysis device is amass spectrometer and the chemical analysis is mass spectrometry. 24.The method of claim 22, wherein the surface tension of the solventcomposition when in a liquid junction with the membrane having ajunction fluid diameter prevents expansion of the junction fluiddiameter beyond two times the diameter of the distal end of the captureprobe.
 25. A method for sampling a sample liquid, comprising the stepsof: providing the sample liquid in a sample fluid conduit, the samplefluid conduit comprising a membrane comprising pores, the membranepreventing the passage of the sample liquid through the pores at a firstpressure of the sample liquid in the sample fluid conduit; flowingsolvent composition from the open end of a solvent supply conduit tocontact the membrane; establishing a second pressure within the solventin contact with the membrane through a solvent exhaust conduit, thesecond pressure being lower than the first pressure; drawing sampleliquid through the pores of the membrane by the pressure differential ofthe first pressure and the second pressure, wherein the extracted sampleliquid is combined with the flowing solvent composition and flows intothe open end of a solvent exhaust conduit.
 26. The method of claim 25,wherein the solvent supply conduit and the solvent exhaust conduit areprovided by varying the pressure within a single tube having an openend.
 27. The method of claim 26, wherein the solvent forms a liquidmicrojunction between the tube and the membrane.
 28. The method of claim25, wherein the solvent is deposited on the membrane by the solventsupply conduit, then the supply of solvent is stopped and the solvent onthe membrane is allowed to stand for a period of time, and then thesecond pressure is applied to the solvent in contact with the membraneby the solvent exhaust conduit.